Stroke has characteristics of high mortality, high disability rate, high recurrence rate, etc. and is seriously harmful to human health. Due to the complexity of the fine structure of brain tissue, it is particularly sensitive and vulnerable to hypoxic-ischemic injuries. So far, therapeutic drugs with definite efficacy are rare in clinical practice. Studies have shown that the excessive release of excitatory amino acids such as glutamate under a condition of cerebral ischemia results in overstimulation of N-methyl-D-aspartate receptor (NMDAR), thereby nitric oxide (NO) is released pathologically through NMDAR-PSD-95-nNOS signaling pathway (Science, 1999, 284, 1845-1848; Nature Medicine 2010, 16, 1439-1443), in which nNOS is neuronal nitric oxide synthase and PSD-95 is postsynaptic density protein-95. NO with a strong oxidizing property itself will directly damage surface lipids and internal structures of the surrounding cells when NO is excessively released or cleared up insufficiently. On the other hand, great amount of oxygen anion free radicals (O2−.) produced during the cerebral ischemia-reperfusion react with NO to generate nitrite superoxide anion free radicals (ONOO−.), which as a strong and stable oxidant will cause more severe damage to cells (Cell. Mol. Life. Sci., 2004, 61, 657-668). Thus, NMDAR-mediated activation of nNOS is a key event to the excitotoxic occurrence of neurons. Although a number of drugs were developed based on the two target molecules, direct intervention with them often leads to severe side effects due to the very important physiological functions of NMDAR and nNOS (Curr. Opin. Pharmacol., 2006, 6, 53-60). Selectively blocking the interaction of NMDAR with PSD-95 (Science, 2002, 298, 846-850) or nNOS with PSD-95 (Nature Medicine 2010, 16, 1439-1443) other than directly interfering NMDAR or nNOS may inhibit pathological release of NO after cerebral ischemia without affecting physiological functions of NMDAR and nNOS, raising the possibility of obtaining safe and effective therapeutic drugs against cerebral ischemia with no significant side effects. Moreover, the coupling of nNOS and PSD-95 is more ideal drug target as it is downstream in the whole signal pathway (Neuropharmacology, 2003, 45, 738-754).
The benzylaniline derivative 4-N-(2-hydroxy-3,5-dichlorobenzyl) aminosalicylic acid (ZL006), an nNOS-PSD-95 uncoupler, reduce NMDAR-mediated pathological NO release, display significant neuroprotective effect against glutamate-induced neuronal injury, and improve animal neurological deficits and reduce infarct volume caused by middle cerebral artery occlusion (MCAO) and reperfusion, without affecting the physiological functions of NMDAR and nNOS (Nature Medicine 2010, 16, 1439-1443). Moreover, ZL006 does not cause learning and memory impairment, behavioral abnormalities and other side effects that may be caused by directly interfering NMDAR or/and nNOS. Thus ZL006 is safer and is of great significance to the treatment of cerebral ischemic injury-related diseases (Nature. Rev. Neurol., 2011, 7, 61).
Pain is an unpleasant feeling and emotional experience caused by tissue damage or potential tissue damage. Under normal physiological conditions, “pain is an alarm signal that warns the body is under threat”, and is also a special protective function indispensable to the body. However, under pathological conditions, tissue damage (including neurogenic and inflammatory) can lead to hyperexcitability of spinal dorsal horn neurons, resulting in central sensitization and generally manifesting as hyperalgesia and allodynia clinically. Recent studies suggest that the balance between excitatory neurotransmitter receptor system and inhibitory neurotransmitter receptor system in the central nervous system (CNS) plays an important role in the maintenance of normal pain threshold.
A large number of experimental results have shown that glutamate is an important excitatory neurotransmitter in the process of nociceptive information transmission in the central nervous system, and it mainly acts on the ionotropic glutamate receptors including non-NMDA receptors and NMDA receptors, and metabotropic glutamate receptors. Wherein, NMDA receptors play a very important role in hyperalgesia process induced by peripheral tissue injury or nerve damage (Nature, 1983, 306, 686-688). Studies have shown that intrathecal injection of MK-801, a noncompetitive antagonist of NMDA receptor, can significantly alleviate mechanical allodynia and thermal hyperalgesia in capsaicin-induced mechanical and thermal hyperalgesia (Neuropharmacology, 2005, 48, 325˜332). Intramuscular injection of AP-5, a competitive antagonist of NMDA receptor, can reduce complete Freund's adjuvant (CFA)-induced muscle hyperalgesia in a dose-dependent manner (Neuroreport, 2005, 16, 485˜489). However, in view of the broad distribution and the extensive function of NMDA receptors in the central nervous system, the use of NMDA receptor antagonists will result in central toxic side effects in many aspects such as memory loss, insanity, and ataxia, which greatly limits the clinical application of NMDA receptor antagonists as analgesics. Therefore, it has become a new target for analgesic drug research by interfering the transmission of pain-related downstream signaling mediated by NMDA receptors, without affecting the function as a channel of the NMDA receptor itself. nNOS is an important signaling molecule in the NMDA receptor signaling pathway. nNOS can be selectively activated by NMDA receptor-mediated Ca2+ influx. In addition to the direct binding to the NMDA receptor, PDZ-2 in PSD-95 interacts with the PDZ domain of nNOS, forming an NMDA receptor/PSD-95/nNOS ternary complex, which provides a platform for the selective activation of nNOS by NMDA receptors-mediated Ca2+. Experiments have shown that interference with the coupling of PSD-95 and nNOS by small molecule compounds and polypeptides similar to the terminal of PSD-95 can significantly reduce the acute thermal hyperalgesia and chronic mechanical hyperalgesia in rodents (Journal of Pain Research, 2011, 158, 494-506). The pain models in rodents show that the nNOS-PSD-95 uncoupler, 2-[(1H-benzotriazole-5-amino)methyl]-4,6-dichlorophenol (IC87201), has significant inhibitory effects against mechanical hyperalgesia and thermal hyperalgesia, and may be developed as an analgesic drug with brand new mechanism of action (Brit. J. Pharmacol., 2009, 158, 494-506). PSD-95 and nNOS uncoupling therefore provide a new approach for screening selective analgesic drugs.
The target of nNOS-PSD-95 uncoupler represented by ZL006 is in the central nervous system, and thus this type of drugs need a good distribution within the central nervous system. However, since this type of uncouplers have high hydrophilicity and are not adapted to oral absorption and distribution within the central nervous system, it is necessary to increase the lipophilicity of the molecules. The present inventors have found that new molecules formed by the esterification or amidation of hydrophilic groups in nNOS-PSD-95 uncoupler represented by ZL006 molecule, administered by the various routes of administration including parenteral administration, have good protective effects against cerebral ischemia and analgesic effects.